Ischemia/reperfusion (I/R) injury contributes significantly to morbidity and mortality in patients with ischemic heart disease. Paradoxically, reperfusion of ischemic tissue causes further myocardial injury, limiting current therapeutic modalities. The goals of this proposal are to examine at a molecular level the signaling pathways that are activated in endothelial cells and cardiomyocytes as a response to the oxidative stress of I/R, and to translate these findings to in vivo models of I/R injury and, ultimately, clinical conditions where I/R injury is encountered. Related to the phenomenon of I/R injury is the observation that short I/R events precondition the heart to tolerate longer I/R events at a later time. Therefore, a more complete understanding of ischemic preconditioning could lead to the development of drugs that precondition the myocardium and make it more resistant to reperfusion after revascularization procedures. Studies performed during the previous award period of this grant have identified Toll-like receptor-4 (TLR4) as a receptor that plays a rote in cardiac damage in a mouse model of I/R injury. To expand upon these observation, three specific aims are proposed that examine TLR4 activation, TLR4 signal transduction and TLR4-mediated transcriptional activation of TNF-alpha expression in cardiac myocytes: (1) Determine the mechanism of TLR4 activation during myocardial ischemia-reperfusion injury and ischemic preconditioning, (2) Determine the mechanism of TLR4 signal transduction in myocardial ischemia-reperfusion injury and ischemic preconditioning, (3) Determine TLR4-mediated transcription activation during myocardial ischemia reperfusion injury. Results generated from the experiments described in this proposal will be applicable to the many clinical settings in which I/R injury is the principal pathogenetic event, such as stroke peripheral vascular disease, hemorrhagic shock and early transplant graft dysfunction. Also, trauma often results in shock (whole-body ischemia) which is treated by rapid transfusion (reperfusion). We anticipate that concepts generated from the studies proposed here will also be applicable to this endemic disease. Our intention is to focus on the role TLR4 and its signaling pathways in I/R injury to more precisely define therapeutic targets that are specific to myocardial I/R injury.